Articles Polar body-based preimplantation diagnosis for X-linked disorders

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1 RBMOnline - Vol 4. No Reproductive BioMedicine Online; on web 20 November 2001 Articles Polar body-based preimplantation diagnosis for X-linked disorders Dr Yury Verlinsky is a graduate, postgraduate and PhD of Kharkov University of the former USSR. His research interests include cytogenetics, embryology and prenatal and preimplantation genetics. He introduced polar body testing for preimplantation genetic diagnosis and developed the methods for karyotyping second polar body and individual blastomeres. He has published over 100 papers, as well as three books on preimplantation genetics. Dr Yury Verlinsky Y Verlinsky 1, 3, S Rechitsky 1, O Verlinsky 1, D Kenigsberg 2, J Moshella 2, V Ivakhnenko 1, C Masciangelo 1, C Strom 1, A Kuliev 1 1 Reproductive Genetics Institute, Chicago, IL, USA 2 Long Island IVF, Port Jefferson, NY, USA 3 Correspondence: 2825 North Halsted Street, Chicago, IL 60657; Tel: (773) ; Fax: (773) ; rgi@flash.net Abstract Preimplantation diagnosis for X-linked disorders has been performed predominantly by gender determination, which, however, leads to the discarding of 50% unaffected male embryos. In an attempt to identify X-linked mutation-free embryos for transfer, the present authors introduced preimplantation genetic diagnosis (PGD), using a sequential first and second polar body analysis, as an alternative to gender determination. This method was offered to eight couples at risk for having children with X-linked disorders, including haemophilia B, fragile-x syndrome (FMR1), myotubular myotonic dystrophy (MTMD), ornithine transcarbamylase (OTC) deficiency and X-linked hydrocephalus. The first and second polar bodies were removed following maturation and fertilization of oocytes in a standard IVF protocol and analysed using a multiplex nested polymerase chain reaction (PCR), involving testing for mutations simultaneously with linked markers. Overall, 13 PGD cycles were performed, resulting in the detection of 25 embryos with the predicted mutation-free maternal contribution; these embryos were transferred back to the patients in all cycles, yielding four clinical pregnancies. Four children were born following these pregnancies, including three unaffected and one with misdiagnosis as a result of allele dropout (ADO), which was predictable in the case of FMR1. Presented results demonstrate the clinical usefulness of the specific polar body testing for X-linked disorders as an alternative to PGD by gender determination. 38 Keywords: first and second polar bodies, linked polymorphic markers, misdiagnosis, multiplex nested polymerase chain reaction, preimplantation genetic diagnosis, X-linked disorders Introduction One of the first cases of preimplantation genetic diagnosis (PGD) was attempted for X-linked disorders more than 11 years ago and has been performed by gender determination (Handyside et al., 1990). The X-linked disorders are currently one of the major indications for PGD, still performed mainly by gender determination (European Society of Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium, 2000; International Working Group on Preimplantation Genetics, 2001a). Gender determination was initially done by single cell polymerase chain reaction (PCR) analysis, while now it is performed by the fluorescence in-situ hybridization (FISH) technique in interphase cells. Although sufficiently accurate, this approach leads to a discarding of 50% unaffected male embryos, which cannot be accepted by many patients. Thus, there has been a need for specific genetic tests allowing the identification of the unaffected healthy male embryos for transfer as an option to preselect the X-linked mutation-free embryos, irrespective of gender. In an attempt to preselect the X-linked mutation-free embryos for transfer, the present authors introduced sequential first and second polar body (PB1and PB2) analysis (Verlinsky et al., 1997, 1999) as an alternative to gender determination. The

2 approach was applied in a group of couples at risk for having children with X-linked genetic disorders. One such case, involving PGD for ornithine transcarbamylase (OTC) deficiency, was reported previously, showing the feasibility of the approach (Verlinsky et al., 2000). This paper describes current experience in the application of PB testing for PGD in a group of eight couples at risk for producing a child with X-linked disorders. Materials and methods Eight couples presented for PGD in connection with their previous male offspring being affected by an X-linked disorder, including haemophilia B (one couple), fragile-x syndrome (FMR1) (three couples), myotubular myotonic dystrophy (MTMD) (one couple), OTC deficiency (one couple) and X- linked hydrocephalus (two couples). Overall, 13 PGD cycles were performed for these couples, one for haemophilia B, four for FMR1, one for MTMD, two for OTC deficiency and five for X-linked hydrocephalus (Table 1). Following maturation of oocytes in a standard IVF protocol, PB1s were removed at least 3 h after oocyte retrieval (38 h after human chorionic gonadotrophin (HCG) administration), using micromanipulation procedures described elsewhere (Verlinsky and Kuliev, 2000). Then oocytes were fertilized by intracytoplasmic sperm injection (ICSI), followed by PB2 removal, approximately h after ICSI, using the micromanipulation techniques mentioned. Although PB1 and PB2 were removed in sequence, they were amplified at the same time, using nested multiplex PCR (Rechitsky et al., 1999), with the primer designs worked out for each of the disorders mentioned above. Primers and PCR conditions for these disorders, except for OTC deficiency described previously (Verlinsky et al., 2000), are presented in Table 2. Based on the sequential PB1 and PB2 testing, involving the mutation and/or linked marker analysis (Rechitsky et al., 1999, 2000), results of which were available already on day 1 (approximately h after oocyte retrieval), the embryos resulting from the oocytes predicted to be free from maternal mutation were transferred back to the patients on day 3, while those predicted to be affected were further tested for confirmation of diagnosis when available. Results and discussion A total of 100 oocytes obtained from 13 cycles (7.7 oocytes per cycle, on average) were with PB1 results, which appeared to be heterozygous in 66 (66%) and homozygous normal or mutant in 34 (34%) oocytes. PB2 results were available in 79 (79%) of these oocytes (6.1 oocytes per cycle, on average), so in the remaining 21 oocytes, the genotype prediction was not possible. Overall, six cases of allele drop out (ADO) were detected. ADO remains the major problem in avoiding misdiagnosis if undetected, as observed in one of the cases of PGD for FMR1, presented below. These six ADO were detected either by the use of the linked markers (two ADO) or by sequential PB1 and PB2 analysis (four ADO), as shown in the case of PGD for X- linked hydrocephalus (Figure 1). As seen from this case, both Figure 1. Sequential first polar body (PB1) and second polar body (PB2) analysis in preimplantation genetic diagnosis (PGD) for X-linked hydrocephalus. Top: localization of frame shift (Fr) mutation 168 in exon 5 and linked short tandem (TCTA) and dinucleotide (CA) repeats. Horizontal arrows represent primer sets for nested polymerase chain reaction (PCR); vertical arrows represent restriction sites for HpaII or polymorphic markers. Middle: Restriction map for Fr 168 mutation analysis. Bottom: Sequential PB1 and PB2 analysis, allowing preselection of oocytes 3 and 4 for embryo transfer (ET), based on heterozygote PB1 and mutant PB2. Additionally, oocyte 8 may also be predicted normal, based on homozygous mutant genotype of both PB1 and PB2, which can be explained only by allele dropout (ADO) of normal allele in heterozygous PB1. Oocytes 5, 7 and 10 (heterozygous PB1 and normal PB2), and oocyte 12 (normal PB1 and mutant PB2) are mutant. Table 1. Polar body analysis for X-linked disorders. Mutation Patient No. oocytes No. embryos Pregnancies /cycle PB1 PB1 + transferred /births PB2 FMR1 3/ /2 MTMD 1/ a OTC deficiency 1/ /2 Hydrocephalus 2/ Haemophilia B 1/ Total 8/ /4 PB1 = results of first polar body analysis; PB2 = results of second polar body analysis; FMR1 = fragile-x syndrome; MTMD = myotubular myotonic dystrophy; OTC = ornithine transcarbamylase. a Chemical 39

3 40 PB1 and PB2 in oocyte 8 had an identical homozygous mutant genotype, suggesting that PB1 was apparently heterozygous, with the normal allele not detected because of ADO. In five PGD cycles performed for this disorder, a total of 12 unaffected embryos were preselected for transfer (at least two embryos in each cycle). These embryos originated from the mutation-free oocytes preselected from 47 oocytes tested (Table 1), based on the heterozygous status of PB1 and homozygous mutant PB2, which not only predicted the mutation-free status of the resulting embryos but also reliably excluded any probability of ADO (oocytes 3 and 4 shown in Figure 1). Unfortunately, none of these transfers yielded a clinical pregnancy. In a single PGD cycle for MTMD, in which seven oocytes were tested by both PB1 and PB2, two unaffected embryos were transferred, one originating from the oocytes with heterozygous PB1 and homozygous mutant PB2, and one with homozygous mutant PB1 and normal PB2, in which ADO in PB1 was excluded by two linked markers, but only chemical pregnancy was obtained. In the other single PGD cycle for haemophilia B, only two oocytes were available for testing by both PB1 and PB2, from which one with heterozygous PB1 and mutant PB2 was predicted normal and transferred, yielding no clinical pregnancy. In four PGD cycles performed for FMR1, the embryos for transfer were preselected in each of them. As the expanded allele for this condition cannot be amplified directly, the preselection of the mutation-free oocytes was inferred from the presence of the linked markers for the normal allele. Overall, 14 of 18 oocytes available were tested by both PB1 and PB2, suggesting the unaffected status in eight of them, based on heterozygous PB1 and mutant PB2 in six, and homozygous mutant PB1 and normal PB2 in two. Of eight embryos transferred in four clinical cycles (three in one, one in one, and two in each of the remaining two), all but two originated from oocytes with heterozygous PB1. Two of these transfers yielded clinical pregnancies, one resulting in the birth of an unaffected child and the other in the birth of an affected child (Figure 2). The first one originated from the transfer of two embryos, both originating from the oocytes with heterozygous PB1 and mutant PB2, while the other involved the transfer of three embryos, two deriving from oocytes with heterozygous PB1 and mutant PB2, and the third from the oocyte with presumably homozygous mutant PB1, which in fact turned out to be heterozygous because of an undetected ADO of both alleles linked to the normal gene (Table 3). As only two of four available markers were informative for linkage analysis in this cycle, in the absence of the direct test for the expanded allele the chance of ADO of the normal allele in this case was predicted to be in the range of 5%, based on previous observations described in detail elsewhere (Rechitsky et al., 2000). Of 15 oocytes available for testing in two PGD cycles for OTC deficiency, nine were with the results of both PB1 and PB2, but only a single mutation-free oocyte was preselected in each cycle, one based on heterozygous PB1 and mutant PB2 and the other homozygous mutant PB1 and normal PB2, but with the linked marker information available to confirm the diagnosis. The transfer of a single embryo in each of these cycles, resulting from the latter mutation-free oocytes, yielded clinical pregnancies, both resulting in the birth of an unaffected child (one of these cases was reported earlier by Verlinsky et al., 2000). Overall, 25 embryos resulting from the oocytes predicted to be free from mutation were transferred back to the patients in all 13 cycles (1.9 per cycle), yielding four clinical pregnancies, three of which have resulted in the birth of unaffected children. Only six embryos resulting from mutant oocytes were available for the follow-up analysis, confirming the predicted results of sequential PB1 and PB2 analysis, including five embryos with X-linked hydrocephalus and one with haemophilia B. As shown in Table 1, both PB1 and PB2 results were obtained in 80% of oocytes. This makes the preselection of a sufficient number of embryos for transfer when only few oocytes were available for analysis unrealistic, and may suggest the usefulness of cancelling cycles in such cases. Although this might be considered as one of the disadvantages of the PB approach, the majority of such cases were due to a fertilization failure with no PB2 extruded, so no embryos were available to perform blastomere biopsy, either. According to the most recent review (International Working Group on Preimplantation Genetics, 2001b), approximately 1000 PGD cycles have been performed worldwide for Mendelian disorders, resulting in more than 200 clinical Figure 2. Misdiagnosis in preimplantation genetic diagnosis (PGD) for fragile-x syndrome (FMR1): pedigree with results of haplotype analysis for CGG expansion in FMR1 gene using two linked markers. Top: haplotypes of the mother carrying an expanded allele shown linked to 154 and 245 markers (in bold). Middle: haplotype of two sisters who inherited the expanded allele from their mother, also shown in bold. One of the sisters presented for PGD is shown by the arrow. Bottom: one of the sisters (on the left) has an affected son, who carries only expanded allele (linked to 154 and 245 markers). On the right, an affected child carrying only expanded allele (linked to 154 and 245 markers) was born following PGD, due to the predicted 5% risk of misdiagnosis, resulting from undetected allele dropout (ADO) of the normal allele in PB1 of the corresponding oocyte (see Table 3), from which the transferred embryo derived.

4 Table 2. Primers and polymerase chain reaction (PCR) conditions for preimplantation genetic diagnosis (PGD) of fragile-x syndrome, haemophilia B, X-linked miotubular miopathy and X-linked hydrocephalus. Gene/ Upper primer Lower primer Anneal. temp. polymorphism a FMR1 AC1 Outside C 5 AGCTGCAAAGAGAAACAGACA 3 5 AATATCAGGCCAGGCACA 3 5 GTTGATGCTGAACATCCTTATCG 3 5 Fam GGCTGAGGCATGATGAGAGTC 3 FMR1 AC2 Outside C 5 GCCCTAATCAGATTTCCACA 3 5 GATGCGGTGGCTCAAG 3 5 CAAAAAGAACCCAGATGTGA 3 5 Fam GGGAGGATAGCTCAAGCTC 3 DXs548 Outside C 5 Fam CCTACATCAAAGTCCCAGCA 3 5 AAGCCTGCAACCAAACACTG 3 5 Fam CCTACATCAAAGTCCCAGCA 3 5 GTACATTAGAGTCACCTGTGGTGC 3 DXs1193 Outside C 5 GCCAAGGCATAGAAGACAAC 3 5 TGTCAGCACAAAAGGGCTTA 3 5 ATTGTTCATGCAACTCTCCTC 3 5 Hex TCATCCAAGCTACTTATTTTAAG 3 Factor IX RFLP Outside 55 C MseI 5 AGAGGGATAAATACATCAATGGC 3 5 AATATATTGTCTCCAGCCTGTAGC 3 5 GATAGAGAAACTGGAAGTAGACCC 3 5 ATTAGGTCTTTCACAGAGTAGAATTT 3 Factor IX RFLP Outside 55 C Mnl I 5 TTGTAATACATGTTCCATTTGCC 3 5 GGGAATTGACCTGGTTTGG 3 5 TTCTAGTGCCATTTCCATGTG 3 5 ATCTTCTCCACCAACAACCC 3 Factor IX VNTR Outside 55 C (50 bp repeat) 5 GGGACCACTGTCGTATAATGTG 3 5 GAAGAGACACTCCTGAACTCTGG 3 5 CCAAAATGTCATTGTGCAGC 3 5 TCTGAATCATATTTCTCCTTCCC 3 MTM1 R241C Outside 55 C (loses artificial 5 TTGGATGTGGTGCTAATTAAG 3 5 CTCATCATCTTTATTTCGTTTC 3 HhaI site) Inside 55 C 5 CGCAGTGAGATTGCAAGTG 3 5 CAAGAGGCTGACTGCA G C 3 MTM1 DXS1684 Outside 55 C 5 AGCACCCAGTAAGAGACTG 3 5 TGAATCAATCTATCCATCTCTC 3 Inside 55 C 5 CAGGCCACTACCACTTATG 3 5 HexTACTGTTTTCCACTCTAATGC 3 X-hydrocephalus Outside 55 C Fr.168 (-C) 5 GAGTGTCAGCCCGTCTG 3 5 AGTAGAGGTTGCCGTTCTG 3 (loses Hpa II site) 5 GTGGCCAAAGGAGACAGTGAA 3 5 GAAGACACCCCCGCTAACA 3 a Explanation for abbreviations is available at 41

5 42 Table 3. Preimplantation genetic diagnosis (PGD) for the fragile-x syndrome (FMR1) gene by linkage analysis in case of misdiagnosis. Oocyte Cell DXs1193 DXs548 Predicted no. type (in bp) (in bp) oocyte genotype 1 PB1 154 a / /247 Affected 3 PB1 154/ /247 Affected 5 PB1 154/ /247 Normal PB PB1 154/ /247 Normal PB PB Normal b 10 PB1 154/ /247 Affected Cord Affected blood PB1 = first polar body; PB2 = second polar body; bp = size of polymerase chain reaction (PCR) product in base pairs a Values in bold indicate markers linked to the expanded allele b Potential 5% risk for allele drop out (ADO) pregnancies. More than half these cycles have been performed for X-linked disorders, by gender determination (ESHRE Preimplantation Genetic Diagnosis Consortium, 2000; International Working Group on Preimplantation Genetics, 2001a). On the one hand, this could be due to the lack of sequence information in many X-linked conditions, although it is also technically easier to identify female embryos by DNA analysis or FISH, despite an obvious cost of discarding 50% healthy male embryos. On the other hand, testing for X-linked genetic disorders might be entirely limited to oocytes, because of the maternal origin of the mutations involved, making useless any further manipulation and testing of the resulting embryos, which may be transferred irrespective of gender or any contribution from the father. The present authors have previously reported the feasibility of such an approach in the case of PGD for OTC deficiency, resulting in the birth of an unaffected child (Verlinsky et al., 2000). A similar case has recently been reported, but this was performed by blastomere biopsy (Ray et al., 2000). Presented results further demonstrate the clinical usefulness of PGD for X-linked disorders by sequential PB1 and PB2 analysis, which resulted in the transfer of the embryos originating from the oocytes predicted to be free from X-linked mutations in all 13 clinical cycles. As mentioned, these 13 transfers yielded four clinical pregnancies, resulting in the birth of three healthy children and one affected child. The three embryos transferred in the latter case included only one originating from the oocyte with the homozygous mutant PB1, predicted on the basis of only one additional polymorphic marker, which leaves a 5% chance for misdiagnosis (Rechitsky et al., 2000). With two embryos having been reliably diagnosed for transfer, the couple might have accepted such a risk of misdiagnosis in order to have three, instead of two, embryos for transfer, improving the woman s chances of becoming pregnant. The results demonstrate once again that the embryos resulting from the oocytes with homozygous mutant PB1should not be transferred unless at least three linked markers are available to exclude the possible heterozygous status of PB1. This is especially relevant to testing for FMR1, which is entirely based on linkage analysis. To exclude the risk for misdiagnosis in the embryos resulting from homozygous affected PB2 completely, testing for as many as four linked markers, overall, is required. In conclusion, the data show that sequential PB1 and PB2 analysis could be a practical option for PGD of X-linked disorders, providing an alternative to PGD by gender determination. References ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium 2000 Data Collecttion II Human Reproduction 15, Handyside AH, Kontogianni EH, Hardy K, Winston RML 1990 Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 344, International Working Group on Preimplantation Genetics 2001a 10th Anniversary of Preimplantation Genetic Diagnosis. Report of the 10th Annual Meeting International Working Group on Preimlantation Genetics, in conjunction with 3rd International Symposium on Preimplantation Genetics, Bologna, June 23, Journal of Assisted Reproduction and Genetics 18, International Working Group on Preimplantation Genetics 2001b Preimplantation Genetic Diagnosis Experience of Three Thousand Clinical Cycles. Report of the 11th Annual Meeting International Working Group on Preimplantation Genetics, in association with 10th International Congress of Human Genetics, Vienna, May 15, Reproductive BioMedicine Online 3, Ray PF, Gigarel N, Bonnefont JP et al First specific preimplantation genetic diagnosis for ornithine transcarbamilase deficiency. Prenatal Diagnosis 20, Rechitsky S, Strom C, Verlinsky O et al Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. Journal of Assisted Reproduction and Genetics 16, Rechitsky S, Verlinsky O, Strom C et al Experience with single-cell PCR in preimplantation genetic diagnosis: how to avoid pitfalls. In: Hahn S, Holzgreve W (eds) Fetal Cells in Maternal Blood. New Developments for a New Millennium. 11th Fetal Cell Workshop, Basel. Karger, Basel, Switzerland, pp Verlinsky Y, Kuliev A 2000 Atlas of Preimplantation Genetic Diagnosis. Parthenon, NY and London. Verlinsky Y, Rechitsky S, Cieslak J et al Preimplantation diagnosis of single gene disorders by two-step oocyte analysis using first and second polar pody. Biochemical and Molecular Medicine 62, Verlinsky Y, Rechitsky S, Verlinsky O et al Prepregnancy testing for single-gene disorders by polar body analysis. Genetic Testing 3, Verlinsky Y, Rechitsky S, Verlinsky O et al PGD for ornithine transcarbamilase deficiency. Reproductive BioMedicine Online 1,